1. PCR assay of the molecular alternations of Drosophila melanogaster cinnabar gene after γ ray 60 Co action Dr Igor Donatovich Alexandrov Liliana Namolovan Genetic Group Laboratory of Nuclear Problems Joint Institute for Nuclear Research Kateřina Šteklová Lenka Tománková, CTU in Prague Tereza Kulatá, CTU in Prague Piotr Kwaśniewski, AMU in Poznan Pavel Bláha, CTU in Prague Pavel Solný, CTU in Prague JINR Summer Student Practices 2010, Dubna

2. Project aim To study the molecular genetic action of gamma rays ( 60 Co) on the cinnabar mutant of Drosophila melanogaster

3. Cinnabar gene structure and... kynurenine-3-hydroxylase Cinnabar gene encodes kynurenine-3-hydroxylase which catalyses reaction : kunurenine ----> 3-hydroxykynurenine

4. … D. melanogaster cinnabar mutant phenotype Cn (cinnabar) mutant WT (wild type) HOMOZYGOTIC MUTANTS CAN`T PRODUCE FUNCTIONAL FORM OF ENZYME – RESULT ON PHENOTYPE LEVEL : BRIGHT RED EYE

5. Main steps

6. DNA isolation - cell lysis - DNA sorbtion with Silica Solution - DNA purification using washing buffer - DNA extraction using Extra Gene

7. DNA structure Sugar-phosphate backbone

8. P olymerase C hain R eaction - PCR - to amplify specific fragment of DNA - method based on in vitro replication of DNA using: Taq polymerase primers dNTP reaction buffer

9. Electrophoresis !

10. Visualization - using ethidium bromide UV UV red-orange